Table Of ContentVOLUME 1—VOLUME V
Edited by
W. H. SEBRELL, JR. and ROBERT S. HARRIS
VOLUME VI and VOLUME VII
Edited by
PAUL GYÖRGY and W. N. PEARSON
THE VITAMINS
Chemistry, Physiology, Pathology, Methods
SECOND EDITION
VOLUME VI
Edited by
PAUL GYORGY W. N. PEARSON
Philadelphia General Hospital Department of Biochemistry
Philadelphia, Pennsylvania School of Medicine
Vanderbilt University
Nashville, Tennessee
ACADEMIC PRESS New York and London
1967
COPYRIGHT © 1967, BY ACADEMIC PRESS INC.
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LIBRARY OF CONGRESS CATALOG CARD NUMBER : 66-26845
PRINTED IN THE UNITED STATES OF AMERICA
Contributors to Volume VI
Numbers in parentheses indicate the pages on which the authors' contributions
begin.
C. I. BLISS (1, 21, 197, 304), The Connecticut Agricultural Experiment
Station and Yale University, New Haven, Connecticut
RAYMOND HOWARD BUNNELL (261), Food and Agricultural Product
Development, Hoffman-La Roche, Inc., Nutley, New Jersey
HENRIK DAM (245), Department of Biochemistry and Nutrition, Poly-
technic Institute, Copenhagen, Denmark
PAUL GYÖRGY (1, 304), Philadelphia General Hospital, Philadelphia,
Pennsylvania
E. KODICEK (211), Dunn Nutritional Laboratory, University of Cam-
bridge, and Medical Research Council, Cambridge, England
D. E. LAWSON (211), Dunn Nutritional Laboratory, University of
Cambridge, and Medical Research Council, Cambridge, England
S. MAHADEVAN (139), Institute of Nutrition Sciences, School of Public
Health and Administrative Medicine, Columbia University, New
York, New York
OSWALD A. ROELS (139, 197), Marine Biology Division, Lamont Ob-
servatory, Columbia University, Palisades, New York
EBBE S0NDERGAARD (245), Department of Biochemistry and Nutrition,
Polytechnic Institute, Copenhagen, Denmark
COLIN WHITE (21), The Connecticut Agricultural Experiment Station
and Yale University, New Haven, Connecticut
v
Preface
The first edition of "Vitamin Methods" edited by P. György ap-
peared more than 15 years ago. Shortly thereafter, the three-volume
treatise entitled "The Vitamins" edited by H. Sebrell and R. Harris
appeared and filled the gap that occurred in the literature at that time.
When it developed that both publications were scheduled for revision
at approximately the same time it seemed appropriate that "Vitamin
Methods" should become part of this comprehensive work.
The appearance of "Vitamin Methods" as Volumes VI and VII of this
edition necessitated certain changes both in format and in subject
matter. The overall effect is that of a new publication rather than a
revision of the first edition. The presentation of the material is organ-
ized around the various vitamins with the physical, chemical, micro-
biological, and animal assays for each vitamin being discussed in a
single chapter. This change reduced much of the overlapping seen in the
first edition, and, we trust, will make this one more convenient to use.
Another innovation—insertion of the references as page footnotes—
gives the reader easy access to the literature without the annoyance of
having to refer constantly to the end of the chapter.
The shifts in technology that have occurred during the past 20 years
are evident. Intermingled with the standard procedures that were
methods of choice 20 years ago and which are still methods of choice
today, the reader will find new Chromatographie procedures, enzymatic
methods, and isotope dilution techniques. Some of the latter are suitable
for routine analyses while others are principally research techniques.
Some of the other techniques described here have not yet had the
benefit of testing in many laboratories. They have been included because
of their potential value in special analytical situations. Although in
many cases continued emphasis on animal tests seemed justified, many
of them detailed in the first edition have been eliminated. Included are
those animal assays which, in the opinion of the authors, were con-
sidered "best." The chapter on statistical treatment of assay data was
retained from the previous edition but in a re-edited form. The
editors and authors invite comments, suggestions, and recommendations,
particularly in regard to the new methods.
Finally, the cooperation and patience of the contributors and the
publishers should not go unrecognized.
August, 1967 Paul György
W. N. Pearson
vii
Contents of Other Volumes
Volume I—Edited by W. H. Sebrell, Jr. and Robert S. Harris
Vitamins A and Carotene Ascorbic Acid
J. Ganguly G. C. Chatterjee
Robert S. Harris Robert S. Harris
0. Isler G. W. Hay
H. Kläui B. A. Lewis
D. McLaren L. W. Mapson
Thomas Moore Mamie Olliver
S. K. Murthy F. Smith
Oswald A. Roels Richard W. Vilter
U. Schwieter
U. Solms
Volume II—Edited by W. H. Sebrell, Jr. and Robert S. Harris
Vitamins B Vitamin B
G V2
G. Brubacher Η· Α· Barker
Paul György Μ· Ε· Coates
Robert S. Harris Karl Folkers
Stanton A. Harris Robert S. Harris
George R. Honig Harold w· Moore
H. Meder D· Perlman
P. Reusser Ε· Η· Reisner
H. E. Sauberlich Harold S. Rosenthal
Klaus Unna Η· Μ· Wuest
F Weber Biotin
H. Weiser _, . _ ..
Oswald Wiss Gyorgy
Robert S. Harris
Bernhardt W. Langer, Jr.
XI
xn CONTENTS OF OTHER VOLUMES
Volume III—Edited by W. H. Sebrell, Jr. and Robert S. Harris
Choline Inositols
Wendell H. Griffith R. J. Anderson
Robert S. Harris S. J. Angyal
W. Stanley Hartroft T. J. Cunha
Joseph F. Nyc Robert S. Harris
Henry A. Lardy
Vitamin D Group
Arthur H. Livermore
Donald Gribetz
Ade T. Milhorat
Robert S. Harris
E. R. Weidlein, Jr,
Harold E. Harrison
James H. Jones
Benjamin Kramer
Juan M. Navia
Milton L. Scott Vitamin K Group
Essential Fatty Acids
H. J. Almquist
George A. Emerson Robert S. Harris
Ralph T. Holman Otto Isler
Joseph J. Rahm Charles A. Owen, Jr.
Hilda F. Wiese Oswald Wiss
Volume IV—Edited by W. H. Sebrell, Jr. and Robert S. Harris
Niacin Sanford A. Miller
Juan M. Navia
C. Gopalan
Paul M. Newberne
Robert S. Harris
Elaine P. Ralli
W. A. Krehl
0. Neal Miller
Pteroylglutamic Acid
Pantothenic Acid
George M. Briggs Robert S. Harris
Alice M. Copping A. Leonard Luhby
Robert S. Harris E. L. R. Stokstad
CONTENTS OF OTHER VOLUMES xiii
Volume V—Edited by W. H. Sebrell, Jr. and Robert S. Harris
Riboflavin Tocopherols
Robert S. Harris Stanley R. Ames
M. K. Horwitt J. Green
Theodor Wagner-Jauregg Robert S. Harris
M. K. Horwitt
Thiamine
Otto Isler
Gene M. Brown Peter Schudel
Robert S. Harris Oswald Wiss
Merton P. Lamden
V. Ramalingaswami Other Growth Factors
Edward F. Rogers
W. H. Sebrell, Jr. G. S. Fraenkel
Klaus Unna R. A. Morton
H. M. Wuest Vernon H. Cheldelin
Volume VII—Edited by Paul György and W. N. Pearson
Principles of Microbiological Assay/W. N. Pearson
Ascorbic Acid/Joseph H. Roe
Thiamine/W. N. Pearson
Riboflavin/W. N. Pearson
Niacin/Grace A. Goldsmith and 0. Neal Miller
Vitamin Ββ/Howerde Säuberlich
Pantothenic Acid/Orson D. Bird and Robert Q. Thompson
Folic Acid/Victor Herbert and Joseph R. Bertino
Vitamin B^/Helen R. Skeggs
Biotin/Paul György
Clinical Evaluation of Malnutrition/Willard A. Krehl
CHAPTER 1
ANIMAL ASSAYS FOR VITAMINS
C I. BLISS AND PAUL GYÖRGY
Introduction 1
I. Precision and Its Measurement 2
II. Basal Rations 4
A. Major Constituents 5
B. Vitamin Supplements 7
C. Salt Mixtures 10
III. Responses Used in Animal Assays for Vitamins 11
A. Growth Assays for Vitamins 12
B. Vitamin Assays Based upon Reaction Time 17
C. Other Graded Response Assays 17
D. All-or-None Vitamin Assays 18
Introduction
Assays with animals have been indispensable for the development of
our present knowledge of the vitamins. Often their purpose has been to
test whether particular preparations could cure deficiency symptoms. In
the isolation of individual vitamins from natural sources, much research
has been guided by the results of such animal assays. As the final cri-
terion for identification of vitamins, animal assays are not likely to be
superseded by microbiological techniques and even less by physical and
chemical methods of analysis. For purposes of control or for routine clin-
ical-chemical studies, however, assays with animals have been largely
replaced by microbiological or chemical and physical techniques.
Despite their importance in research and for validating alternate meth-
ods of assay, the animal assays for the vitamins have been needlessly
inexact. One objective of the present chapter is to emphasize the methods
that lead to quantitative estimates of potency and of their reliability.
Precautions developed for some vitamins seem to have been overlooked in
similar assays of other vitamins, so that many opportunities exist for
improving the assay of one vitamin from knowledge gained with another.
Some of these possibilities are suggested in the present chapter. The design
1
2 1. ANIMAL ASSAYS FOR VITAMINS
and statistical analysis of vitamin assays are developed systematically
in Chapter 2 on "Statistical Methods in Biological Assay of the Vitamins."
For general aspects of small animal experimentation such as breeding of
animals, establishing animal laboratories, etc., see Guerrant.1
Several methods have been developed for assay of each vitamin, each
with its own advantages and limitations. The technical skills required
differ widely. A laboratory that has the necessary equipment and experi-
ence may prefer an assay that is inherently more complex but makes use
of its existing facilities and personnel. The animal assays for a given
vitamin are included in the chapter dealing with the vitamin in question.
They have been restricted to the most commonly used and most reliable
techniques.
I. Precision and Its Measurement
There are two approaches to the problem of ensuring reproducible
results from animal biological assays. One is to specify its exact conduct
in meticulous detail. This implies that if everyone were to follow exactly
the same procedure, each should arrive at the same result. If in fact a
given technique measures the vitamin activity of an unknown preparation,
an alternative technique for measuring the same activity should lead to the
same answer. If not, one would question the validity of one or both methods
or the applicability of the results of either to a different species. A more
realistic approach is to allow greater flexibility in experimental detail,
but to determine the standard error or confidence limits of each deter-
mination as an integral part of the assay. A method that works well in
one laboratory may be quite unsatisfactory in another, but its exact details
are of little consequence if we know the inherent precision of an estimated
potency and the general method by which it was obtained.
Much of the precision of an assay is determined by its design and also
by the method of evaluating the results. A basic principle is to administer
at least one vitamin preparation in each assay at two or more dosage
levels, in order to provide a quantitative estimate of the change in response
that corresponds to a known change in dose. A second principle is that
an assay must be comparative, with concurrent observations on the test
material or unknown and on the reference preparation or standard. Given
these two prerequisites, the activity of the unknown can be expressed in
terms of an equivalent quantity of the standard rather than of units of
response. Living material is subject to so many variations, both inherent
and environmental, that considerable ingenuity may be required to keep
1 N. B. Guerrant, in "Vitamin Methods" (P. György, ed.), Vol. 2, pp. 1-40. Aca-
demic Press, New York, 1951.
